Introduction To Spectral Cytometry Using Aurora And .
Introduction to Spectral Cytometry using Cytek Aurora and Northern LightsDay 3
What happens when I have too much caffeine!
A B cell panel for human PBMCsMarkerFluorLevel of Expression /Cell FrequencyControl: Beadsor BV785BV605Pacific BlueBV650B711High/ intermediateHigh/ intermediateIntermediate/ highHigh/ highHigh/intermediateHigh/ intermediateLow/ lowCellsCellsCellsCellsCellsCellsBeadsPBMCsControl Stained WithAny bright BV510Any bright BV421CD27 BV785, same lotCD62L BV605, same lotAny bright Pacific BlueIgM BV650, same lotIgG BV711, same lot
Intracellular Cytokine Staining/Proliferation Assay,cultured PBMCsMarkerFluorCFSECD3PE CF594CD4APCCD8APC R700IFNgefluor 450IL-4PE Cy7IL-17PerCP e710viability Aqua Amine DyeunstainedLevel of Expression /Cell FrequencyControl: Beadsor CellsControl Stained WithHigh/highLow/ highLow/highIntermediate/highIntermediate/ int.High/lowLow/lowhigh/lowUnstim cellsUnstim cellsUnstim cellsCellsStim cellsStim cellsBeadsCellsPBMCs culturedCFSE well titrated*CD3 PE-CF594, same lotAny bright APC, but CD4 is goodCD8 APC-R700, same lotAny bright eFluor 450IL-4 PE-Cy7, same lot, higher stopping gate for enough eventsIL-17 PerCPeFluor710, same lotAqua Amine DyeUnstim and stimulatedSeparate controls*Switch CFSE to Cell Trace Violet – much easier to titrate into range
You are planning to perform an assay for detection of FOXP3 and to follow the protocol below. Please write downthe protocol you will follow (step by step) to stain beads to be used as controls for surface antigens AND forintracellular antigensCells1. Add surface Abs2.Incubate/wash3.Fix Perm4.Wash5.AF647 Ab6.Incubate/wash7.Run!Beads for Surface AgsBeads for i.c. Ags
Reference Control QCPE control:GoodPositive peak SpectrumNegative peak Spectrum
Reference Control QCPE control:neg peak has pos signal – ALWAYS WASH YOUR BEADS/CELLS
Reference Control QCSuper Bright 780 Control: not highest peak gated, highest peak has incorrect signature (has SB436 sig) tandemdegradation, pos peak in bottom plot is the donor, the acceptor is no longer fluorescing
Reference Control QCBB700 Control: Pos gate too wide, too heterogeneous, plot 2 and 3 show different spectra, lower peak looks like carryover
Reference Control QCPE-Cy5 Control: a lot of tailing on pos peak – usually when beads get stuck on the side of the tube and encountervariable amounts of Ab, this is also a problem with old beads that don’t bind uniformly
Knowledge Review Analyze full spectrum signatures Understand how full spectrum signatures are generated Identify unique signatures Normalize spectrum signatures QC and troubleshoot signatures Understand the fundamentals of spectral unmixing How it works How it compares to compensation Understand the workflow in Spectroflo Understand the requirements for optimal reference controls How to QC reference controls
ObjectivesBy the end of Day 3, you should be able to: Understand how to plan and design a multicolor panel Understand the fundamentals of panel design Understand the tools available for fluorochrome selection and successful paneldesign Panel performance evaluation and optimization strategies
Know the BiologyPlanning a Multicolor Experiment
Panel Design: Gathering InformationIdentify Antigens Level of expression: primary, secondary, tertiary Antigen co-expressionSelect Fluorochromes Unique signature Brightness Spread considerationsCheck Availability Are the reagents available in the antibody/fluor combination
Antigen ClassificationPrimaryHigh densityon or off expressionSecondaryCD3CD4CD8Intermediate densitycontinuous expressionCD45RACCR7CD127HLA-DRTertiaryLow densityunknown expressionPD-1CD25TCR g/dFlurochrome BrightnessLevel of Antigen ExpressionCD4CD127CD25Mahnke, Y. and Roederer, M. Clin Lab Med. 2007 September ; 27(3): 469
Know your co-expression
Selecting Fluorochromes with Unique SignaturesSeveral tools available tohelp:1. Fluorochrome selectionguidelines2. Online spectrum viewer3. Similarity & complexityindices
Laser UniqueLook at andBlueIdentifyUniqueSignaturesdyes to use
Fluorochrome selectionStain index
Assessing Brightness: Stain Index Ranking of 65 DyesStain IndexDimMidBrightVery Bright
Example: 35 Fluorochrome SelectionApprox. EmissionWavelength 30750780800UVVioletBlueYellow GreenRedBUV395BUV496BV421Super Bright 436eFluor 450BV480BV510BUV563Pacific OrangeBV570BUV661BV605BV650Live/Dead CP-Cy5.5PerCP-eFluor710PE-Cy5APCAlexa Fluor 647APC-R700BUV737BUV805BV750BV785Qdot 800PE-Cy7APC-Fire 750
Fluorochrome selectionUnderstanding Spread
Assessing Antigen ResolutionMULTICOLOR SCENARIOMarker A Fluor XSINGLE STAINEDCountsMarker A Fluor XCountsSINGLE COLOR SCENARIOMFI Fluor XMarker A Fluor XMULTICOLOR TUBECountsMarker A Fluor YMFI Fluor YMain Contributors for Resolution Reduction Instrument Performance Instrument Setup Fluorochrome BrightnessCountsMFI Fluor XMFI Fluor XMain Contributors for Resolution Reduction Spread Antibody titer
Quantification of Impact of Spread in ResolutionMFI Fluor YMarker A Fluor XStain IndexMarker A Fluor XFluor X DOES NOTspread into Fluor YFluor Y DOES NOTspread into Fluor XMFI Fluor YMULTICOLOR SCENARIOSINGLE COLOR SCENARIOStain IndexMarker A Fluor YMFI Fluor XMFI Fluor XFluor X DOESSPREAD into Fluor YFluor Y DOES NOTspread into Fluor XMFI Fluor YMarker A Fluor YMFI Fluor YMFI Fluor XStain Indices UnchangedStain Index Marker A Fluor YDECREASES in presence of fluor XMFI Fluor XConsiderations CO EXPRESSION ANTIGEN LEVEL OF EXPRESSION Data used for calculations has to be unmixed using a certain combination of fluorochromes
Fluor X DOESSPREAD into Fluor YFluor Y DOES NOTspread into Fluor XMFI Fluor YCross Stain Index Calculation1Stain Index Marker A Fluor Y2 DECREASES in presence of fluor XMFI Fluor XBy unmixing CD4 stained cellswith multiple fluors we canassess how much the positivepopulation spread into otherdyes (rSD) .Spread % ReductionWe use that spread to calculate how much thesingle stained Stain Index (1) is reduced by thespread introduced by another dye (2)25
Cross Stain Index Matrix: 35 Color PanelBUV395LIVE DEAD BlueBUV496BUV563BUV661BUV737BUV805BV421Super Bright 436eFluor 450BV480BV510Pacific OrangeBV570BV605BV650BV711BV750BV785Qdot CF568PE-Dazzle594PE-Cy5PE-Cy7APCAlexa Fluor 647APC-R700APC-eFluor 780Dye in row impacts dye in columnNoImpactHighImpactUnmixed CD4 PE-Cy7BUV805Unmixed CD4 APCBUV395BUV395LIVE DEAD BlueBUV496BUV563BUV661BUV737BUV805BV421Super Bright 436eFluor 450BV480BV510Pacific OrangeBV570BV605BV650BV711BV750BV785Qdot 800BB515FITCCF514PerCPPerCP-Cy5.5PerCP-eFluor 710PECF568PE-Dazzle594PE-Cy5PE-Cy7APCAlexa Fluor 647APC-R700APC-eFluor 780APCPE-Cy7
35-Color PanelApprox. EmissionWavelength 30750780800UVVioletBlueYellow GreenRedCD45RA BUV395CD16 BUV496PD-1 BV421CD123 SB436CD161 eFluor 450IgD BV480CD3 BV510CD14 BUV563CD20 Pacific OrangeHLA DR BV570CD11c BUV661CD28 BV605CXCR3 BV650Live/Dead BlueCCR6 BV711CD86 BB515CD57 FITCCD19 CF514CD335 PECD4 CF568CD24 PE Dazzle594CD45 PerCPCD95 PE-Cy5CD11b PerCP Cy5.5TCR γδ PerCP eFluor710CD27 APCCD33 Alexa647CD127 APC-R700CD56 BUV737CD45RO BUV805CXCR5 BV750CCR7 BV785CD8 Qdot 800PrimaryCD25 PE Cy7SecondaryTertiaryCD38 APC-eFluor 780
Panel Design and Highly Overlapping Dyes (1)TCRgd
May 05, 2020 · 700 APC-780 BUV395 LIVE DEAD Blue BUV496 BUV563 BUV661 BUV737 BUV805 BV421 Super Bright 436 eFluor 450 BV480 BV510 Pacific Orange BV570 BV605 BV650 BV711 BV750 BV785 Qdot 800 BB515 FITC CF514 PerCP PerCP-Cy5.5 PerCP-eFluor 710 PE CF568 PE-Dazzle594 PE-Cy5 PE-Cy7 APC Alexa Fluor 647 APC-R700 APC-eFluor 780 No Impact High Impact Unmixed
Introduction to Flow Cytometry: A Learning Guide 6 Figure 2-1 Hydrodynamic focusing of the sample core through a flow cell Figure 2-2 Hydrodynamic focusing of the sample core through a nozzle tip The sample pressure and the sheath fluid pressure are different from each other. The sample pressure is always greater than the sheath fluid pressure.File Size: 1MBPage Count: 58Explore furtherFlow Cytometry Basics Guide - Bio-Radwww.bio-rad-antibodies.comInterpreting flow cytometry data: a guide for the ter 4: Data Analysis Flow Cytometry - A Basic .flowbook.denovosoftware.comHow to Understand Flow Cytometry Results Sciencingsciencing.comFlow Cytometry Fundamental Principle - Bosterbiowww.bosterbio.comRecommended to you b
Practical Flow Cytometry. 3rd ed. New York, NY: Alan R. Liss; 1995 (ISBN -471-30376-3). Introduction to Flow Cytometry: A Learning Guide vi. 1 1 Overview Flow cytometry is a technology that simultaneously measures and then analyzes multiple physical characteristics of single particles, usually cells, as they flow in a fluid .
diagnosed through the detection of deficient antigens on red blood cells, monocytes, and/or granulocytes by flow cytometry.1 Criteria Introduction Requests for flow cytometry testing are reviewed using these criteria. This guideline addresses common clinical applications of flow cytometry-based te
flow cytometry in research and its possibilities to be used in the clinic. Keywords: flow cytometry; multiparameter analysis; immunophenotyping 1. Introduction Flow cytometry first invented in the late 1960s is an extremely versatile and powerful tool for the quantitative and qualitative analysis of cells on a single-cell level [1]. While
For Multicolour analysis: Panel design: Deciding on the panel Fluorochrome selection and spacing to avoid spectral overlap . Wash/lyse/fix sequence evaluation Gating strategy . Challenges of Flow Cytometry Method Validation Accuracy: For Flow Cytometry - Establishing accuracy in an assay is often impractical - as the true value .
Keywords: flow cytometry, automated analysis, gating, high-throughput, software, immunology. introduction. Recent advances in flow cytometry (FCM) have provided researchers in the fields of cellular and clinical immunology an incredible amount of leverage toward testing new hypotheses. These include
speech enhancement techniques, DFT-based transforms domain techniques have been widely spread in the form of spectral subtraction [1]. Even though the algorithm has very . spectral subtraction using scaling factor and spectral floor tries to reduce the spectral excursions for improving speech quality. This proposed
Power Spectral Subtraction which itself creates a bi-product named as synthetic noise[1]. A significant improvement to spectral subtraction with over subtraction noise given by Berouti [2] is Non -Linear Spectral subtraction. Ephraim and Malah proposed spectral subtraction with MMSE using a gain function based on priori and posteriori SNRs [3 .
